Antidiuretic hormone (ADH) plays a key role in regulating water balance by controlling renal water transport. ADH binds to the vasopressin-2 receptor (V2R) and increases expression of aquaporin 2 (AQP2) water channels in collecting duct cells to increase renal tubular water permeability. A steep solute corticomedullary gradient in the medullary interstitium provides the driving force for water absorption from tubular fluid in the distal nephron. We and others have demonstrated that ADH also stimulates activity of the epithelial sodium channel (ENaC) in the collecting duct and that ENaC possibly facilitates urine concentration. However, ENaC is traditionally regarded as the end-effector of the renin-angiotensin-aldosterone system (RAAS) and the final element that controls renal Na+ excretion and thus blood pressure. Yet we have shown that in some cases of hyponatremia, such as after adrenalectomy, ENaC activity is surprisingly robust. After adrenalectomy, serum ADH levels rise, leading to an increase in ADH-mediated ENaC activity. This increase in ENaC activity occurs in the absence of aldosterone, raising many new questions about ADH regulation of ENaC. The notion that ENaC can respond to both ADH and RAAS is at odds with the accepted role of this channel in merely controlling renal Na+ reabsorption and blood pressure. To reconcile the apparent conflicting roles of ADH in regulating both water and balance, we propose a unifying paradigm that defines the contribution of ENaC as a regulator of water and Na+ balance. We hypothesize that simultaneous activation of ENaC and AQP2 by ADH promotes urine concentration and plasma dilution. If parallel signaling pathways stimulate ENaC activity to a sufficiently high level, then ADH-mediated ENaC activation will induce Na+ retention beyond what is needed for urine concentration and lead to hypertension. We will use state-of-the-art methodologies (e.g., patch clamp studies of isolated split-open collecting duct in mice) and novel reagents (connecting segment/collecting duct-specific Nedd4-2 knockout mice) to test three Specific Aims: 1) Test whether ENaC contributes to pathologic renal water reabsorption and hyponatremia, 2) Test whether V2R activation can induce renal Na+ retention and high blood pressure, and 3) Test whether V2R signals through Nedd4-2 to stimulate ENaC in vivo. This proposal will provide mechanistic insights into how ADH, AQP2, and ENaC interact and ultimately control water and Na+ homeostasis. This knowledge is clinically significant because it may suggest new strategies, which are already currently available to clinicians, for the treatment of hyponatremia and hypertension.